Angiogenesis is a complex multi-step process that can occur in development and diseases with a vascular component in response to various stimuli. Many lines of evidence indicate that angiogenesis depends on proteolytic activity. Activated blood vessels express biochemical markers that are differentially expressed and whose functional importance tias just begun to be uncovered. While only a few markers associated with angiogenic blood vessels have thus far been reported, it is remarkable that several are cell membrane-associated proteinases. By using genetic elimination and Diochemical inhibition, we have uncovered an as yet unrecognized mechanistic role for APA and CD13/APN in pathological angiogenesis. We hypothesize that APA and CD 13 activity contribute to important regulatory pathways. Here, we propose to investigate the mechanisms by which CD 13 and APA expression and activity control the physiological and pathological proliferation of activated endothelial cells forming blood vessels. First, we will study the induction and activity of CD 13 and APA during angiogenesis. We will evaluate the expression of CD 13 and of APA in angiogenic vasculature. As cell surface, membrane-bound enzymes upregulated in tumor vessels, APA and CD 13 may contribute to the initiation and/or progression of angiogenesis by (i) degrading inhibitory peptides, (ii) activating stimulatory molecules, or both. We will establish whether or not the levels of peptides known to bind and be degraded by APA and CD 13 are altered in activated blood vessels. Our main focus will be on the interdependent substrates for these enzymes, angiotensin III and VI. Angiotensin III has been shown to be pro-angiogenic, supporting our hypothesis. Second, we will determine the nature of the angiogenic stimuli leading to upregulation and activation of CD13/APN and APA in endothelial cells and pericytes. We will also examine the effects of inhibitors of CD13/APN and APA in angiogenesis. We will utilize anti-CD 13 and anti-APA inhibitory antibodies and peptides to evaluate if these peptidases enhance/accelerate angiogenesis ex-vivo and in vivo. Finally, we will evaluate the phenotype of CD13 and APA deficient mice with respect to angiogenesis using cytokine-, oxygen-and tumor-induced angiogenesis models. It is our hypothesis that these peptidases play complementary roles in angiogenesis. Our studies are likely to establish a mechanistic basis for the role of pro-angiogenic peptidases in the formation and maintenance of neovasculature, an aspect of high relevance in vascular biology. The proposed experiments in this application may also lead to development of new therapeutic strategies for diseases with an angiogenic component such as cancer and retinopathies.